APIP (also known as MtnB) is a zinc-dependent methylthioribulose-1-phosphate dehydratase that catalyzes the dehydration of MTRu-1-P to 2,3-diketo-5-methylthiopentyl-1-phosphate in the methionine salvage pathway. This is its primary evolved enzymatic function. The protein also has a moonlighting function as an inhibitor of apoptosis through competitive binding to Apaf-1, which is independent of its enzymatic activity. Additionally, APIP inhibits caspase-1-dependent pyroptosis in an enzyme-activity-dependent manner. The protein exists as a homotetramer and is localized in the cytoplasm.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | ACCEPT | Summary: APIP is localized to the cytoplasm. Immunofluorescence studies showed "APIP was detected mainly in the cytoplasm of HeLa cells" (PMID:23285211). The IBA annotation from phylogenetic inference is consistent with experimental evidence. Reason: Cytoplasmic localization is well-supported by immunofluorescence data (PMID:23285211) and is consistent with its role in the cytosolic methionine salvage pathway. Supporting Evidence: PMID:23285211 In accordance with its putative role in the methionine salvage pathway, APIP was detected mainly in the cytoplasm of HeLa cells by immunofluorescence |
| GO:0019509 L-methionine salvage from methylthioadenosine | IBA GO_REF:0000033 | ACCEPT | Summary: APIP functions as MtnB in the methionine salvage pathway. This is its primary enzymatic function. Knockdown studies showed that "APIP depletion specifically impaired the capacity of cells to grow" when methionine was replaced by MTA (PMID:23285211). Ko et al. (2012) confirmed "The role of APIP in methionine salvage was confirmed by growth assays with methionine-deficient media and quantitation of the methionine salvage substrate, 5'-methylthioadenosine" (PMID:22837397). Reason: This represents the core evolved function of APIP. Multiple studies demonstrate APIP's essential role in the methionine salvage pathway through functional assays. Supporting Evidence: PMID:23285211 We show that APIP depletion specifically impaired the capacity of cells to grow PMID:22837397 The role of APIP in methionine salvage was confirmed by growth assays with methionine-deficient media and quantitation of the methionine salvage substrate, 5'-methylthioadenosine |
| GO:0046570 methylthioribulose 1-phosphate dehydratase activity | IBA GO_REF:0000033 | ACCEPT | Summary: APIP catalyzes the dehydration of MTRu-1-P with measured kinetic parameters of Km ~9.32 uM and Vmax ~1.39 umol/min/mg (PMID:24367089). The crystal structure at 2.0 A resolution (PDB 4M6R) confirms a class II aldolase fold with active-site zinc. This is the core molecular function of APIP. Reason: This is the primary molecular function of APIP, well-characterized structurally and biochemically. Supporting Evidence: PMID:24367089 Here we report the structural and enzymatic characterization of human APIP as an MtnB enzyme with a Km of 9.32 ΞΌM and a Vmax of 1.39 ΞΌmol min(-1) mg(-1) |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | ACCEPT | Summary: IEA annotation for cytoplasm based on InterPro domains and UniProt subcellular location data. Consistent with experimental IDA evidence from PMID:23285211. Reason: Consistent with experimental evidence; duplicates IBA and IDA annotations but this is acceptable as it represents an independent computational prediction that agrees with experimental data. Supporting Evidence: PMID:23285211 APIP was detected mainly in the cytoplasm of HeLa cells by immunofluorescence |
| GO:0006915 apoptotic process | IEA GO_REF:0000043 | MARK AS OVER ANNOTATED | Summary: This annotation is an OVER-ANNOTATION. APIP does not participate in apoptosis - it INHIBITS apoptosis as a moonlighting function separate from its enzymatic activity. Kang et al. (2014) demonstrated that "APIP/MtnB functions as a cell death inhibitor independently of its MtnB enzyme activity for apoptosis induced by either hypoxia or etoposide" (PMID:24367089). The gene was named "APAF1-interacting protein" because it binds Apaf-1 CARD and competes with procaspase-9, thereby inhibiting apoptosome formation. GO:0006915 (apoptotic process) implies direct participation in apoptosis, which is inappropriate for an inhibitor. Reason: APIP is an INHIBITOR of apoptosis, not a participant. The IEA mapping from UniProt keyword "Apoptosis" does not distinguish between proteins that participate in apoptosis and those that regulate/inhibit it. The proper annotation would be GO:0043066 (negative regulation of apoptotic process), which is already annotated with IMP evidence. Supporting Evidence: PMID:24367089 APIP/MtnB functions as a cell death inhibitor independently of its MtnB enzyme activity for apoptosis induced by either hypoxia or etoposide PMID:24367089 APIP, Apaf-1 interacting protein, has been known to inhibit two main types of programmed cell death, apoptosis and pyroptosis file:human/APIP/APIP-deep-research-falcon.md APIP competes with procaspase-9 for Apaf-1 binding, inhibiting apoptosome-driven caspase-9 activation and offering cytoprotection in hypoxia/ischemia models |
| GO:0008270 zinc ion binding | IEA GO_REF:0000104 | ACCEPT | Summary: APIP is a zinc-dependent enzyme. The crystal structure (PDB 4M6R) shows zinc coordination by His115, His117, and His195 (PMID:24367089). UniProt notes "Binds 1 zinc ion per subunit." IEA is consistent with IDA evidence. Reason: Zinc binding is essential for catalytic activity and is demonstrated in the crystal structure. The IEA annotation is consistent with experimental IDA evidence. Supporting Evidence: PMID:24367089 an overall fold similar to members of the zinc-dependent class II aldolase family |
| GO:0008652 amino acid biosynthetic process | IEA GO_REF:0000043 | ACCEPT | Summary: The methionine salvage pathway regenerates methionine from 5'-methylthioadenosine. While technically methionine biosynthesis, GO:0019509 (L-methionine salvage from methylthioadenosine) is more specific and appropriate. This general term is acceptable but less informative. Reason: APIP does participate in amino acid biosynthesis (methionine salvage), though the more specific term GO:0019509 is preferred and already annotated. Supporting Evidence: PMID:23285211 these results confirm the involvement of APIP in the methionine salvage pathway |
| GO:0016829 lyase activity | IEA GO_REF:0000043 | ACCEPT | Summary: APIP has EC 4.2.1.109 (lyase activity). The more specific term GO:0046570 (methylthioribulose 1-phosphate dehydratase activity) is already annotated with IDA evidence. This general lyase annotation is acceptable but less informative. Reason: APIP is indeed a lyase (EC 4.2.1.109). The annotation is correct but the more specific GO:0046570 is preferred and already present. Supporting Evidence: PMID:24367089 5-methylthioribulose-1-phosphate dehydratase, or MtnB, in the methionine salvage pathway |
| GO:0019509 L-methionine salvage from methylthioadenosine | IEA GO_REF:0000120 | ACCEPT | Summary: IEA annotation for methionine salvage based on InterPro domains. Consistent with IBA and IMP evidence for this annotation. Reason: This is the core biological process for APIP. The IEA annotation from domain analysis agrees with experimental evidence. Supporting Evidence: PMID:23285211 these results confirm the involvement of APIP in the methionine salvage pathway |
| GO:0046570 methylthioribulose 1-phosphate dehydratase activity | IEA GO_REF:0000120 | ACCEPT | Summary: IEA annotation for the dehydratase activity based on Rhea reaction mapping (EC:4.2.1.109). Consistent with IDA evidence from PMID:24367089. Reason: This is the core molecular function of APIP. The computational annotation agrees with direct enzyme assay data. Supporting Evidence: PMID:24367089 Here we report the structural and enzymatic characterization of human APIP as an MtnB enzyme with a Km of 9.32 ΞΌM and a Vmax of 1.39 ΞΌmol min(-1) mg(-1) |
| GO:0046872 metal ion binding | IEA GO_REF:0000120 | ACCEPT | Summary: APIP binds zinc as a cofactor. The more specific GO:0008270 (zinc ion binding) is already annotated. This general metal ion binding annotation is correct but less informative. Reason: APIP binds zinc ions. The annotation is correct, though the more specific zinc ion binding term is preferred. Supporting Evidence: PMID:24367089 zinc-dependent class II aldolase family |
| GO:0005515 protein binding | IPI PMID:16189514 Towards a proteome-scale map of the human protein-protein in... | REMOVE | Summary: High-throughput protein-protein interaction study. "Protein binding" is uninformative and should generally be avoided per curation guidelines. Reason: "Protein binding" does not tell us about the actual function of APIP. The interaction detected is likely part of high-throughput screening and does not represent a specific functional interaction. More informative terms like "identical protein binding" for homomeric interactions are preferred. Supporting Evidence: PMID:16189514 Towards a proteome-scale map of the human protein-protein interaction network. |
| GO:0005515 protein binding | IPI PMID:19060904 An empirical framework for binary interactome mapping. | REMOVE | Summary: High-throughput interactome mapping study. Uninformative term. Reason: "Protein binding" is too vague and uninformative about APIP's actual function. Supporting Evidence: PMID:19060904 An empirical framework for binary interactome mapping. |
| GO:0005515 protein binding | IPI PMID:25416956 A proteome-scale map of the human interactome network. | REMOVE | Summary: Proteome-scale interactome mapping. Generic protein binding annotation. Reason: "Protein binding" is uninformative. High-throughput interaction data should be represented by more specific binding terms where possible. Supporting Evidence: PMID:25416956 A proteome-scale map of the human interactome network. |
| GO:0005515 protein binding | IPI PMID:27107014 An inter-species protein-protein interaction network across ... | REMOVE | Summary: Inter-species protein-protein interaction study. Generic annotation. Reason: Uninformative term from high-throughput study. Supporting Evidence: PMID:27107014 An inter-species protein-protein interaction network across vast evolutionary distance. |
| GO:0005515 protein binding | IPI PMID:29892012 An interactome perturbation framework prioritizes damaging m... | REMOVE | Summary: High-throughput interactome perturbation study. Generic annotation. Reason: Uninformative term. Supporting Evidence: PMID:29892012 Jun 11. An interactome perturbation framework prioritizes damaging missense mutations for developmental disorders. |
| GO:0005515 protein binding | IPI PMID:31515488 Extensive disruption of protein interactions by genetic vari... | REMOVE | Summary: Study on genetic variants affecting protein interactions. Generic annotation. Reason: Uninformative term from high-throughput study. Supporting Evidence: PMID:31515488 Extensive disruption of protein interactions by genetic variants across the allele frequency spectrum in human populations. |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | REMOVE | Summary: Reference map of human binary interactome. Generic protein binding annotation. Reason: Uninformative. The identical protein binding annotation (GO:0042802) from the same reference is more informative. Supporting Evidence: PMID:32296183 Apr 8. A reference map of the human binary protein interactome. |
| GO:0005515 protein binding | IPI PMID:32814053 Interactome Mapping Provides a Network of Neurodegenerative ... | REMOVE | Summary: Interactome mapping for neurodegenerative disease proteins. Generic annotation. Reason: Uninformative term. Supporting Evidence: PMID:32814053 Interactome Mapping Provides a Network of Neurodegenerative Disease Proteins and Uncovers Widespread Protein Aggregation in Affected Brains. |
| GO:0005515 protein binding | IPI PMID:33961781 Dual proteome-scale networks reveal cell-specific remodeling... | REMOVE | Summary: Dual proteome-scale networks study. Generic protein binding annotation. Reason: Uninformative term from high-throughput study. Supporting Evidence: PMID:33961781 2021 May 6. Dual proteome-scale networks reveal cell-specific remodeling of the human interactome. |
| GO:0042802 identical protein binding | IPI PMID:16189514 Towards a proteome-scale map of the human protein-protein in... | ACCEPT | Summary: APIP forms homotetramers. The crystal structure (PDB 4M6R) shows "APIP/MtnB exists as a tetramer in solution and exhibits an assembly with C4 symmetry" (PMID:24367089). Identical protein binding is appropriate for homooligomeric proteins. Reason: APIP is a homotetramer, and self-interaction is required for its enzymatic function. The annotation accurately reflects the protein's quaternary structure. Supporting Evidence: PMID:24367089 APIP/MtnB exists as a tetramer in solution and exhibits an assembly with C4 symmetry in the crystal lattice PMID:16189514 Towards a proteome-scale map of the human protein-protein interaction network. |
| GO:0042802 identical protein binding | IPI PMID:19060904 An empirical framework for binary interactome mapping. | ACCEPT | Summary: Duplicate annotation for APIP homotetrameric assembly. Same rationale as above. Reason: Consistent with structural evidence for homotetramer formation. Supporting Evidence: PMID:24367089 APIP/MtnB exists as a tetramer in solution PMID:19060904 An empirical framework for binary interactome mapping. |
| GO:0042802 identical protein binding | IPI PMID:25416956 A proteome-scale map of the human interactome network. | ACCEPT | Summary: Additional evidence for APIP self-interaction/homotetramer formation. Reason: Consistent with structural and biochemical evidence. Supporting Evidence: PMID:24367089 APIP/MtnB exists as a tetramer in solution PMID:25416956 A proteome-scale map of the human interactome network. |
| GO:0042802 identical protein binding | IPI PMID:31515488 Extensive disruption of protein interactions by genetic vari... | ACCEPT | Summary: APIP self-interaction detected in genetic variant study. Reason: Consistent with known homotetrameric structure. Supporting Evidence: PMID:24367089 APIP/MtnB exists as a tetramer in solution PMID:31515488 Extensive disruption of protein interactions by genetic variants across the allele frequency spectrum in human populations. |
| GO:0042802 identical protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | ACCEPT | Summary: APIP self-interaction in binary interactome reference map. Reason: Consistent with known homotetrameric structure. Supporting Evidence: PMID:24367089 APIP/MtnB exists as a tetramer in solution PMID:32296183 Apr 8. A reference map of the human binary protein interactome. |
| GO:0070372 regulation of ERK1 and ERK2 cascade | IEA GO_REF:0000107 | UNDECIDED | Summary: Transferred from mouse ortholog. The evidence for APIP involvement in ERK1/2 cascade regulation is weak and may not represent a core function. Reason: Unable to access primary literature for the mouse ortholog annotation to evaluate the strength of evidence. This may represent a peripheral or context-dependent effect rather than a core function. |
| GO:0005737 cytoplasm | IDA PMID:23285211 Functional identification of APIP as human mtnB, a key enzym... | ACCEPT | Summary: Direct experimental evidence for cytoplasmic localization by immunofluorescence. "APIP was detected mainly in the cytoplasm of HeLa cells" (PMID:23285211). Reason: Strong experimental evidence for cytoplasmic localization, consistent with its role in the cytosolic methionine salvage pathway. Supporting Evidence: PMID:23285211 In accordance with its putative role in the methionine salvage pathway, APIP was detected mainly in the cytoplasm of HeLa cells by immunofluorescence |
| GO:0008270 zinc ion binding | IDA PMID:24367089 Structural and biochemical basis for the inhibition of cell ... | ACCEPT | Summary: Crystal structure at 2.0 A resolution (PDB 4M6R) directly demonstrates zinc binding at the active site. His115, His117, and His195 coordinate the zinc ion. Reason: High-resolution structural data directly demonstrates zinc binding, which is essential for catalytic activity. Supporting Evidence: PMID:24367089 The crystal structure was determined at 2.0-Γ
resolution, revealing an overall fold similar to members of the zinc-dependent class II aldolase family |
| GO:0043066 negative regulation of apoptotic process | IMP PMID:24367089 Structural and biochemical basis for the inhibition of cell ... | KEEP AS NON CORE | Summary: APIP inhibits caspase-9-dependent apoptosis by binding to Apaf-1 CARD and competing with procaspase-9 for apoptosome formation. Importantly, "APIP/MtnB functions as a cell death inhibitor independently of its MtnB enzyme activity for apoptosis" (PMID:24367089). This is a moonlighting function. Reason: This is a well-documented moonlighting function of APIP that is INDEPENDENT of its enzymatic activity. It represents a secondary regulatory role rather than the primary evolved function. The anti-apoptotic effect requires physical binding to Apaf-1 but not dehydratase activity. Supporting Evidence: PMID:24367089 APIP/MtnB functions as a cell death inhibitor independently of its MtnB enzyme activity for apoptosis induced by either hypoxia or etoposide PMID:24367089 APIP, Apaf-1 interacting protein, has been known to inhibit two main types of programmed cell death, apoptosis and pyroptosis |
| GO:0046570 methylthioribulose 1-phosphate dehydratase activity | IDA PMID:24367089 Structural and biochemical basis for the inhibition of cell ... | ACCEPT | Summary: Direct enzyme assay demonstrated dehydratase activity with Km = 9.32 uM and Vmax = 1.39 umol/min/mg. Mutational analysis confirmed active site residues (Q96A, C97A, H115A, E139A affect activity). Reason: This is the core molecular function of APIP, demonstrated by direct biochemical characterization with kinetic parameters. Supporting Evidence: PMID:24367089 Here we report the structural and enzymatic characterization of human APIP as an MtnB enzyme with a Km of 9.32 ΞΌM and a Vmax of 1.39 ΞΌmol min(-1) mg(-1) |
| GO:0051289 protein homotetramerization | IDA PMID:24367089 Structural and biochemical basis for the inhibition of cell ... | ACCEPT | Summary: Crystal structure and solution studies demonstrate APIP forms a homotetramer with C4 symmetry. "APIP/MtnB exists as a tetramer in solution and exhibits an assembly with C4 symmetry in the crystal lattice" (PMID:24367089). Reason: Structural evidence directly demonstrates tetrameric assembly, which is required for enzymatic function (active site is at subunit interface). Supporting Evidence: PMID:24367089 APIP/MtnB exists as a tetramer in solution and exhibits an assembly with C4 symmetry in the crystal lattice. The pocket-shaped active site is located at the end of a long cleft between two adjacent subunits. |
| GO:0070269 pyroptotic inflammatory response | IMP PMID:24367089 Structural and biochemical basis for the inhibition of cell ... | KEEP AS NON CORE | Summary: APIP inhibits caspase-1-dependent pyroptosis. Unlike its anti-apoptotic function, the anti-pyroptotic activity IS dependent on enzymatic activity. Kang et al. showed that enzymatic mutants lose the ability to protect against pyroptosis but retain anti-apoptotic activity (PMID:24367089). Reason: This represents a secondary regulatory function linked to the methionine salvage pathway. The inhibition of pyroptosis requires APIP's enzymatic activity, suggesting metabolic regulation of inflammation. However, this is not the primary function of APIP. Supporting Evidence: PMID:24367089 APIP/MtnB functions as a cell death inhibitor independently of its MtnB enzyme activity for apoptosis induced by either hypoxia or etoposide, but dependently for caspase-1-induced pyroptosis |
| GO:0005829 cytosol | TAS Reactome:R-HSA-6804596 | ACCEPT | Summary: Reactome pathway annotation for APIP binding to the apoptosome complex (APAF1:CYCS). Cytosolic localization is consistent with immunofluorescence data (PMID:23285211). Reason: Cytosolic localization is consistent with experimental evidence and with APIP's role in both cytosolic methionine salvage and apoptosome regulation. Supporting Evidence: PMID:23285211 APIP was detected mainly in the cytoplasm of HeLa cells by immunofluorescence |
| GO:0019509 L-methionine salvage from methylthioadenosine | IMP PMID:22837397 Functional genetic screen of human diversity reveals that a ... | ACCEPT | Summary: Ko et al. (2012) confirmed APIP's role in methionine salvage through growth assays and MTA quantitation. "The role of APIP in methionine salvage was confirmed by growth assays with methionine-deficient media and quantitation of the methionine salvage substrate, 5'-methylthioadenosine" (PMID:22837397). Reason: This is the core biological process of APIP, demonstrated by functional mutant phenotype studies. Supporting Evidence: PMID:22837397 The role of APIP in methionine salvage was confirmed by growth assays with methionine-deficient media and quantitation of the methionine salvage substrate, 5'-methylthioadenosine |
| GO:0019509 L-methionine salvage from methylthioadenosine | IMP PMID:23285211 Functional identification of APIP as human mtnB, a key enzym... | ACCEPT | Summary: Mary et al. (2012) demonstrated that APIP knockdown impairs cell growth when methionine is replaced by MTA. "Stable knockdown of APIP specifically affects growth in MTA and depletes intracellular levels of methionine" (PMID:23285211). Reason: Independent IMP evidence confirming APIP's essential role in methionine salvage pathway. Supporting Evidence: PMID:23285211 We show that APIP depletion specifically impaired the capacity of cells to grow |
| GO:0043066 negative regulation of apoptotic process | IMP PMID:22837397 Functional genetic screen of human diversity reveals that a ... | KEEP AS NON CORE | Summary: Ko et al. showed that reduced APIP expression increases sensitivity to caspase-9-dependent cell death. "Consistent with APIP originally being identified as an inhibitor of caspase-9-dependent apoptosis, the same allele was also associated with increased sensitivity to the chemotherapeutic agent carboplatin" (PMID:22837397). Reason: This is a moonlighting function independent of enzymatic activity. It is a legitimate regulatory role but not the primary evolved function of APIP. Supporting Evidence: PMID:22837397 Consistent with APIP originally being identified as an inhibitor of caspase-9-dependent apoptosis, the same allele was also associated with increased sensitivity to the chemotherapeutic agent carboplatin |
| GO:0070372 regulation of ERK1 and ERK2 cascade | ISS GO_REF:0000024 | UNDECIDED | Summary: Annotation transferred from mouse ortholog Q9WVQ5 by sequence similarity. Reason: Cannot evaluate without access to the primary evidence for the mouse annotation. This may represent a peripheral or context-dependent effect. |
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Download this section (compressed HTML)Q: What is the mechanism by which APIP's enzymatic activity regulates pyroptosis but not apoptosis? The differential requirement for enzymatic activity between anti-apoptotic (enzyme-independent) and anti-pyroptotic (enzyme-dependent) functions suggests distinct regulatory mechanisms.
Q: Does APIP have additional moonlighting functions related to its numerous protein-protein interactions detected in high-throughput studies? APIP interacts with many proteins beyond Apaf-1 and caspase-1, and the functional significance of these interactions is unclear.
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